Abstract

During early B cell development, the stepwise arrangement of the B cell receptor (BCR) by immunoglobulin (Ig) gene recombination defines developmental stages and coordinates checkpoints for differentiation. Early pre-B cells express an incomplete version of the BCR, referred to as the pre-BCR, which signals clonal expansion and additional Ig rearrangements. These cellular programs are essential for B cell development and are carried out in a highly regulated fashion during the pre-B cell checkpoint. Clonal expansion defines the strictly proliferative large pre-B cell stage, while Ig rearrangements occur exclusively in small pre-B cells. Signaling in pre-B cells must carefully orchestrate cessation of pre-BCR signals that drive proliferation while promoting Ig recombination. Ig recombination proceeds through DNA double-stranded breaks, which induce DNA damage responses (DDR) that trigger new cellular programming to counter certain pre-BCR driven signals. Precisely how pre-BCR and DDR signals are coordinated in vivo remains unknown and represents an important mechanism of B cell developmental regulation. This thesis presents data that define the kinetics of pre-BCR and DDR signaling during pre-B cell checkpoint and characterize the heterogeneity of developmental states within small pre-B cells. Using a novel mouse model that encodes a fluorescently tagged SYK (SykmCherry/+), downstream regulator of pre-BCR signaling, we define an inverse relationship between pre-BCR signaling and DDR in small pre-B cells in vivo. Single-cell RNA-sequencing (scRNA-seq) of murine bone marrow B cells defines transcriptionally unique subpopulations of small pre-B cells that transition between high levels of pre-BCR versus DDR signaling. Previous in vitro work established that transcription factors SPIC/BCLAF1, activated by non-canonical DDR (ncDDR) in small pre-B cells, compete with pre-BCR-driven transcription factor PU.1 for DNA binding sites to downregulate Syk expression. Here we find in vivo that BCLAF1 has inverse DNA binding kinetics relative to PU.1 and predominates in stages of small pre-B cells with low SYK expression. These data illustrate the opposing kinetics of pre-BCR and DDR signaling in vivo during pre-B cell stage and the dynamic cellular programming during the pre-B checkpoint. Disrupted signaling during the pre-B checkpoint introduces risk to normal B cell development, such as leukemic transformation. For this reason, we investigated the role of the ncDDR factors SPIC/BCLAF1 in maintaining pre-B checkpoint integrity. Using mouse models with B-cell specific deletion of BCLAF1 we find that that loss of SPIC/BCLAF1 results in an expansion of large pre-B cells and a reduction in small pre-B cells. Additionally, we find that BCLAF1-deficient large pre-B cells show transcriptional and phenotypic similarities to small pre-B cells, suggesting a breakdown in checkpoint stability that leads to a reversal in developmental progression. These ncDDR pathways may provide independent or cooperative signals to preserve pre-B cell checkpoint and prevent malignant transformation of developing B cells. In a longitudinal aging study of mice with B cell specific loss of ncDDR factors SPIC/BCLAF1 alone or in combination with loss of the cDDR factor P53, we find that combined loss of SPIC/BCLAF1 and P53 results in an aberrant B cell expansion that is more likely to show signs of arrest at the pro-B and pre-B cell stages. Taken together, these findings emphasize the importance of ncDDR programming in B cell development and pre-B checkpoint integrity.

Committee Chair

Jeffrey Bednarski

Committee Members

Abby Green; Laura Schuettpelz; Peggy Kendall; Yoon-A Kang

Degree

Doctor of Philosophy (PhD)

Author's Department

Biology & Biomedical Sciences (Immunology)

Author's School

Graduate School of Arts and Sciences

Document Type

Dissertation

Date of Award

8-6-2026

Language

English (en)

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